Antisense modulation of SRA expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the function or amount of SRA. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to SRA or nucleic acids encoding SRA. Methods of using these compounds for modulation of SRA levels and for treatment of diseases associated with SRA are provided.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of SRA. In particular, this invention relates toantisense compounds, particularly oligonucleotides, specificallyhybridizable with SRA or nucleic acids encoding human SRA. Sucholigonucleotides have been shown to modulate the expression of SRA.

BACKGROUND OF THE INVENTION

Steroid, thyroid and retinoid hormones produce a diverse array ofphysiologic effects through the regulation of gene expression. Uponentering the cell, these hormones bind to a unique group ofintracellular nuclear receptors which have been characterized asligand-dependent transcription factors. This complex then moves into thenucleus where the receptor and its cognate ligand interact with thetranscription preinitiation complex affecting its stability andultimately the rate of transcription of the target genes.

The interactions of the liganded receptor with the specific elements inthe promoter region of the target genes are mediated by multi-proteincomplexes. These complexes are composed of either corepressors, whichinhibit transactivation or coactivators, which enhance transactivation(Glass et al., Curr. Opin. Cell. Biol., 1997, 9, 222-232). Severalnuclear receptor coactivator proteins have been identified, all havingsimilar biochemical properties. Recently, however, a novel coactivatorwas reported to be the first endogenous RNA transcript to function as aneukaryotic transcriptional coactivator. This coactivator, SRA, orsteroid receptor RNA activator, has several unique features. SRAselectively interacts with the AF-1 domain of steroid hormone receptorsfound in the amino terminus of the receptor. Nuclear receptors containtwo AF (activation function) domains responsible for their transcriptionactivation function, and previously identified coactivators have allbeen shown to interact through the AF-2 domain.

SRA is also expressed as multiple isoforms in a tissue and cell-specificmanner. Multiple transcripts of SRA were isolated and fell into twocategories; a major transcript fraction of 0.7-0.85 kilobases (kb) inlength producing a doublet and a minor transcript fraction of 1.3-1.5kilobases. Of the two major transcripts in the doublet, the 0.7 kbspecies was expressed at significantly higher levels in breast cancercell lines. All of the transcripts were found to exist at differentlevels in the tissues examined but were enriched in the liver andskeletal muscle.

Finally, SRA was found to be present in a preformed coregulator complexwith the AF-2 coactivator, SRC-1. SRC-1 or steroid receptorcoactivator-1 has been shown to enhance the transcriptional activity ofthe estrogen, glucocorticoid, thyroid, progesterone and retinoic acidreceptors (Onate et al., Science, 1995, 270, 1354-1357). Inimmunoprecipitation experiments, it was shown that SRA acts as acomponent of distinct ribonucleoprotein complexes, one of which containsthe nuclear receptor coactivator, SRC-1. Thus the SRA mRNA transcriptitself, specifically expressed in steroid target tissues, functions as acomponent of a large multiprotein complex to selectively enhancetranscriptional activation by steroid receptors.

Currently, there are no known therapeutic agents that effectivelyinhibit the synthesis of SRA. Consequently, there remains a long feltneed for agents capable of selectively and effectively inhibiting SRAfunction.

Antisense technology is emerging as an effective means for reducing theexpression of specific gene products and may therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications for the modulation of SRA expression.

SUMMARY OF THE INVENTION

The present invention is directed to antisense compounds, particularlyoligonucleotides, which are targeted to SRA or a nucleic acid encodingSRA, and which modulate SRA or the expression of SRA. Pharmaceutical andother compositions comprising the antisense compounds of the inventionare also provided. Further provided are methods of modulating levels ofSRA in cells or tissues comprising contacting said cells or tissues withone or more of the antisense compounds or compositions of the invention.Further provided are methods of treating an animal, particularly ahuman, suspected of having or being prone to a disease or conditionassociated with SRA by administering a therapeutically orprophylactically effective amount of one or more of the antisensecompounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric antisense compounds,particularly oligonucleotides, for use in modulating the function of SRAor nucleic acid molecules encoding SPA, ultimately modulating the amountof SRA present. This is accomplished by providing antisense compoundswhich specifically hybridize with an SRA transcript or nucleic acidsencoding SPA. As used herein, the terms "target nucleic acid" and"nucleic acid encoding SRA" encompass SRA, DNA encoding SPA, RNA(including pre-mRNA and mRNA) transcribed from such DNA, and also cDNAderived from such RNA. The specific hybridization of an oligomericcompound with its target nucleic acid interferes with the normalfunction of the nucleic acid. This modulation of function of a targetnucleic acid by compounds which specifically hybridize to it isgenerally referred to as "antisense" The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, and catalytic activity which may be engaged in orfacilitated by the RNA.

The overall effect of such interference with target nucleic acidfunction is modulation of the expression of SRA. In the context of thepresent invention, "modulation" means either an increase (stimulation)or a decrease (inhibition) in the expression of a gene. In the contextof the present invention, inhibition is the preferred form of modulationof gene expression and mRNA is a preferred target. Because it ispresently believed that SRA may not be translated and the SRA mRNAtranscript itself may be the functional moiety in transcriptionalcoactivation, the target molecule is the SRA mRNA transcript itself,which may then be inactivated or degraded through antisense activity, ora nucleic acid encoding SRA, antisense targeting to which would thenblock SRA expression.

It is preferred to target specific nucleic acids for antisense."Targeting" an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is SRA or anucleic acid molecule encoding SRA. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5'-AUG (in transcribed mRNAmolecules; 5'-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the "AUG codon," the "startcodon" or the "AUG start codon". A minority of genes have a translationinitiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, theterms "translation initiation codon" and "start codon" can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, "start codon" and "translationinitiation codon" refer to the codon or codons, if any, that would beused in vivo to initiate translation of an mRNA molecule transcribedfrom a gene encoding SRA, regardless of the sequence(s) of such codons.

It is also known in the art that a translation termination codon (or"stop codon") of a gene may have one of three sequences, i.e., 5'-UAA,5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAGand 5'-TGA, respectively). The terms "start codon region" and"translation initiation codon region" refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5' or 3') from a translationinitiation codon. Similarly, the terms "stop codon region" and"translation termination codon region" refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5' or 3') from a translationtermination codon.

The open reading frame (ORF) or "coding region," which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Other target regions include the 5' untranslatedregion (5'UTR), known in the art to refer to the portion of an mRNA inthe 5' direction from the translation initiation codon, and thusincluding nucleotides between the 5' cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3' untranslated region (3'UTR), known in the art to refer to theportion of an mRNA in the 3' direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3' end of an mRNA or corresponding nucleotides onthe gene. The 5' cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5'-most residue of the mRNA via a 5'-5'triphosphate linkage. The 5' cap region of an mRNA is considered toinclude the 5' cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5' cap region may also be a preferred targetregion.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as "introns," which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as "exons" and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. It has also been found thatintrons can also be effective, and therefore preferred, target regionsfor antisense compounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect.

In the context of this invention, "hybridization" means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. "Complementary," as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, "specifically hybridizable" and "complementary" are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses.

Antisense oligonucleotides have been employed as therapeutic moieties inthe treatment of disease states in animals and man. Antisenseoligonucleotides have been safely and effectively administered to humansand numerous clinical trials are presently underway. It is thusestablished that oligonucleotides can be useful therapeutic modalitiesthat can be configured to be useful in treatment regimes for treatmentof cells, tissues and animals, especially humans.

In the context of this invention, the term "oligonucleotide" refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. This term includes oligonucleotidescomposed of naturally-occurring nucleobases, sugars and covalentinternucleoside (backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. The antisense compounds in accordance with thisinvention preferably comprise from about 8 to about 30 nucleobases.Particularly preferred are antisense oligonucleotides comprising fromabout 8 to about 30 nucleobases (i.e. from about 8 to about 30 linkednucleosides). As is known in the art, a nucleoside is a base-sugarcombination. The base portion of the nucleoside is normally aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. Nucleotides are nucleosidesthat further include a phosphate group covalently linked to the sugarportion of the nucleoside. For those nucleosides that include apentofuranosyl sugar, the phosphate group can be linked to either the2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides,the phosphate groups covalently link adjacent nucleosides to one anotherto form a linear polymeric compound. In turn the respective ends of thislinear polymeric structure can be further joined to form a circularstructure, however, open linear structures are generally preferred.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3'to 5' phosphodiester linkage.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3'-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3'-5' linkages, 2'-5' linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.:5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular --CH₂ --NH--O--CH₂ --, --CH₂--N(CH₃)--O--CH₂ --, [known as a methylene (methylimino) or MMIbackbone], --CH₂ --O--N(CH₃)--CH₂ --, --CH₂ --N(CH₃)--N(CH₃)--CH₂ -- and--O--N(CH₃)--CH₂ --CH₂ -- [wherein the native phosphodiester backbone isrepresented as --O--P--O--CH₂ --] of the above referenced U.S. Pat. No.5,489,677, and the amide backbones of the above referenced U.S. Pat. No.5,602,240. Also preferred are oligonucleotides having morpholinobackbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2' position: OH; F; O--, S--, or N-alkyl; O--, S--, or N-alkenyl;O--, S-- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyland alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ toC₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n) O]_(m)CH₃, O(CH₂)_(n) OCH₃, O(CH₂)_(n) NH₂, O(CH₂)_(n) CH₃, O(CH₂)_(n) ONH₂,and O(CH₂)_(n) ON[(CH₂)_(n) CH₃)]₂, where n and m are from 1 to about10. Other preferred oligonucleotides comprise one of the following atthe 2' position: C₁ to C₁₀ lower alkyl, substituted lower alkyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN,CF₃, OCF₃, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2'-methoxyethoxy (2'-O--CH₂ CH₂ OCH₃,also known as 2'-O--(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2'-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ ON(CH₃)₂ group, also known as 2'-DMAOE, as described in exampleshereinbelow.

Other preferred modifications include 2'-methoxy (2'-O--CH₃),2'-aminopropoxy (2'-OCH₂ CH₂ CH₂ NH₂) and 2'-fluoro (2'-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3' position of the sugar on the 3'terminal nucleotide or in 2'-5' linked oligonucleotides and the 5'position of 5' terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in theart simply as "base") modifications or substitutions. As used herein,"unmodified" or "natural" nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2'-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205;5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; and 5,681,941, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference, and U.S. Pat. No. 5,750,692, which iscommonly owned with the instant application and also herein incorporatedby reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. "Chimeric"antisense compounds or "chimeras," in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The antisense compounds of the invention are synthesized in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis of antisensemolecules. The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations include,but are not limited to, U.S. Pat. Nos.: 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and pharmaceutically acceptable salts of the compounds ofthe invention, pharmaceutically acceptable salts of such prodrugs, andother bioequivalents.

The term "prodrug" indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 to Imbach et al.

The term "pharmaceutically acceptable salts" refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., "Pharmaceutical Salts," J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a "pharmaceutical addition salt"includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The antisense compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of SRA is treated by administering antisense compounds inaccordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to SRA or nucleic acidsencoding SRA, enabling sandwich and other assays to easily beconstructed to exploit this fact. Hybridization of the antisenseoligonucleotides of the invention with SPA or a nucleic acid encodingSRA can be detected by means known in the art. Such means may includeconjugation of an enzyme to the oligonucleotide, radiolabelling of theoligonucleotide or any other suitable detection means. Kits using suchdetection means for detecting the level of SPA in a sample may also beprepared.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2'-O-methoxyethyl modification are believed to beparticularly useful for oral administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising of two immiscible liquid phases intimately mixed anddispersed with each other. In general, emulsions may be eitherwater-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueousphase is finely divided into and dispersed as minute droplets into abulk oily phase the resulting composition is called a water-in-oil (w/o)emulsion. Alternatively, when an oily phase is finely divided into anddispersed as minute droplets into a bulk aqueous phase the resultingcomposition is called an oil-in-water (o/w) emulsion. Emulsions maycontain additional components in addition to the dispersed phases andthe active drug which may be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation.

Antioxidants used may be free radical scavengers such as tocopherols,alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, orreducing agents such as ascorbic acid and sodium metabisulfite, andantioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of reasons of ease of formulation, efficacyfrom an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories--surfactants, fatty acids,bile salts, chelating agents, and non-chelating non-surfactants (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92).Each of these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term "liposome" means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes. As the mergingof the liposome and cell progresses, the liposomal contents are emptiedinto the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include "sterically stabilized" liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C ₁₂ 15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes. U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the "head") provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p.92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or"surface-active agents") are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term "bile salts"includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, "carrier compound" or"carrier" can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

Excipients

In contrast to a carrier compound, a "pharmaceutical carrier" or"excipient" is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include, but are not limitedto, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin,bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 1206-1228). Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. See, generally, The Merck Manual of Diagnosis and Therapy,15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and46-49, respectively). Other non-antisense chemotherapeutic agents arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Numerous examples of antisensecompounds are known in the art. Two or more combined compounds may beused together or sequentially.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀ s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2'-alkoxy amidites

2'-Deoxy and 2'-methoxy beta-cyanoethyldiisopropyl phosphoramidites werepurchased from commercial sources (e.g. Chemgenes, Needham Mass. or GlenResearch, Inc. Sterling Va.). Other 2'-O-alkoxy substituted nucleosideamidites are prepared as described in U.S. Pat. No. 5,506,351, hereinincorporated by reference. For oligonucleotides synthesized using2'-alkoxy amidites, the standard cycle for unmodified oligonucleotideswas utilized, except the wait step after pulse delivery of tetrazole andbase was increased to 360 seconds.

Oligonucleotides containing 5-methyl-2'-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

2'-Fluoro amidites

2'-Fluorodeoxyadenosine amidites

2'-fluoro oligonucleotides were synthesized as described previously[Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No.5,670,633, herein incorporated by reference. Briefly, the protectednucleoside N6-benzoyl-2'-deoxy-2'-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and by modifying literature procedures whereby the2'-alpha-fluoro atom is introduced by a S_(N) 2-displacement of a2'-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladeninewas selectively protected in moderate yield as the3',5'-ditetrahydropyranyl (THP) intermediate. Deprotection of the THPand N6-benzoyl groups was accomplished using standard methodologies andstandard methods were used to obtain the 5'-dimethoxytrityl-(DMT) and5'-DMT-3'-phosphoramidite intermediates.

2'-Fluorodeoxyguanosine

The synthesis of 2'-deoxy-2'-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5'-DMT- and5'-DMT-3'-phosphoramidites.

2'-Fluorouridine

Synthesis of 2'-deoxy-2'-fluorouridine was accomplished by themodification of a literature procedure in which2,2'-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5'-DMT and 5'-DMT-3'phosphoramidites.

2'-Fluorodeoxycytidine

2'-deoxy-2'-fluorocytidine was synthesized via amination of2'-deoxy-2'-fluorouridine, followed by selective protection to giveN4-benzoyl-2'-deoxy-2'-fluorocytidine. Standard procedures were used toobtain the 5'-DMT and 5'-DMT-3'phosphoramidites.

2'-O-(2-Methoxyethyl) modified amidites

2'-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2'-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid that was crushed to a light tan powder (57 g, 85%crude yield). The NMR spectrum was consistent with the structure,contaminated with phenol as its sodium salt (ca. 5%). The material wasused as is for further reactions (or it can be purified further bycolumn chromatography using a gradient of methanol in ethyl acetate(10-25%) to give a white solid, mp 222-4° C.).

2'-O-Methoxyethyl-5-methyluridine

2,2'-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃ CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂ Cl₂ /acetone/MeOH (20:5:3)containing 0.5% Et₃ NH. The residue was dissolved in CH₂ Cl₂ (250 mL)and adsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct. Additional material was obtained by reworking impure fractions.

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine

2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃ CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂ SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et₃ NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by TLC by first quenching the TLC sample with the addition ofMeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃ CN (700 mL) and set aside. Triethylamine (189 mL,1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH₃ CN (1L), cooled to -5° C. and stirred for 0.5 h using an overhead stirrer.POCl₃ was added dropwise, over a 30 minute period, to the stirredsolution maintained at 0-10° C., and the resulting mixture stirred foran additional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine

A solution of3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄ OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, TLC showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/hexane (1:1) containing 0.5% Et₃ NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine-3'-amidite

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂ Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (TLC showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂ Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2'-O-(Aminooxyethyl) nucleoside amidites and2'-O-(dimethylaminooxyethyl) nucleoside amidites

2'-(Dimethylaminooxyethoxy) nucleoside amidites

2'-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the artas 2'-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.

5'-O-tert-Butyldiphenylsilyl-O² -2'-anhydro-5-methyluridine

O² -2'-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g,0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) weredissolved in dry pyridine (500 ml) at ambient temperature under an argonatmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane(125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. Thereaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22,ethyl acetate) indicated a complete reaction. The solution wasconcentrated under reduced pressure to a thick oil. This was partitionedbetween dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L)and brine (1 L). The organic layer was dried over sodium sulfate andconcentrated under reduced pressure to a thick oil. The oil wasdissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) andthe solution was cooled to -10° C. The resulting crystalline product wascollected by filtration, washed with ethyl ether (3×200 mL) and dried(40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMRwere consistent with pure product.

5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine

In a 2 L stainless steel, unstirred pressure reactor was added borane intetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and withmanual stirring, ethylene glycol (350 mL, excess) was added cautiouslyat first until the evolution of hydrogen gas subsided.5'-O-tert-Butyldiphenylsilyl-O² -2'-anhydro-5-methyluridine (149 g,0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added withmanual stirring. The reactor was sealed and heated in an oil bath untilan internal temperature of 160° C. was reached and then maintained for16 h (pressure<100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine

5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried over P₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂ Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at -10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂ Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂ SO₄. The solution was concentrated to get2'-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%).

5'-O-tert-Butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂ Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂ SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂ SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂ Cl₂ to get5'-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

2'-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dryTHF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). Thismixture of triethylamine-2HF was then added to5'-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂ Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂ Cl₂ to get 2'-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine

2'-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) wasdried over P₂ O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4'-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂ Cl₂ (containing a fewdrops of pyridine) to get5'-O-DMT-2'-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂ O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹ -tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂ SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2'-(Aminooxyethoxy) nucleoside amidites

2'-(Aminooxyethoxy) nucleoside amidites [also known in the art as2'-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

The 2'-O-aminooxyethyl guanosine analog may be obtained by selective2'-O-alkylation of diaminopurine riboside. Multigram quantities ofdiaminopurine riboside may be purchased from Schering AG (Berlin) toprovide 2'-O-(2-ethylacetyl) diaminopurine riboside along with a minoramount of the 3'-O-isomer. 2'-O-(2-ethylacetyl) diaminopurine ribosidemay be resolved and converted to 2'-O-(2-ethylacetyl)guanosine bytreatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D.,Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection proceduresshould afford 2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosineand2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-0-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

Example 2

Oligonucleotide synthesis

Unsubstituted and substituted phosphodiester (P═O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

Phosphinate oligonucleotides are prepared as described in U.S. Pat. No.5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3'-Deoxy-3'-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3

Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 4

PNA Synthesis

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the "gap" segment oflinked nucleosides is positioned between 5' and 3' "wing" segments oflinked nucleosides and a second "open end" type wherein the "gap"segment is located at either the 3' or the 5' terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas "gapmers" or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as "hemimers" or "wingmers".

[2'-O-Me]--[2'-deoxy]--[2'-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate and2'-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2'-deoxy-5'-dimethoxytrityl-3'-O-phosphor-amidite for the DNA portionand 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for 5' and 3'wings. The standard synthesis cycle is modified by increasing the waitstep after the delivery of tetrazole and base to 600 s repeated fourtimes for RNA and twice for 2'-O-methyl. The fully protectedoligonucleotide is cleaved from the support and the phosphate group isdeprotected in 3:1 ammonia/ethanol at room temperature overnight thenlyophilized to dryness. Treatment in methanolic ammonia for 24 hrs atroom temperature is then done to deprotect all bases and sample wasagain lyophilized to dryness. The pellet is resuspended in 1M TBAF inTHF for 24 hrs at room temperature to deprotect the 2' positions. Thereaction is then quenched with 1M TEAA and the sample is then reduced to1/2 volume by rotovac before being desalted on a G25 size exclusioncolumn. The oligo recovered is then analyzed spectrophotometrically foryield and for purity by capillary electrophoresis and by massspectrometry.

[2'-O-(2-Methoxyethyl)]--[2'-deoxy]--[2'-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2'-O-(2-methoxyethyl)]--[2'-deoxy]--[-2'-O-(methoxy-ethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2'-O-methyl chimeric oligonucleotide, with thesubstitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methylamidites.

[2'-O-(2-Methoxyethyl)Phosphodiester]--[2'-deoxyPhosphorothioate]--[2'-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[2'-O-(2-methoxyethyl phosphodiester]--[2'-deoxyphosphorothioate]--[2'-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2'-O-methyl chimeric oligonucleotide with the substitution of2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

Oligonucleotide Isolation

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹ p nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

Oligonucleotide Synthesis--96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a standard 96 well format. Phosphodiesterinternucleotide linkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄ OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

Oligonucleotide Analysis--96 Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing four cell types are provided for illustrative purposes, butother cell types can be routinely used.

T-24 cells:

The transitional cell bladder carcinoma cell line T-24 was obtained fromthe American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cellswere routinely cultured in complete McCoy's 5A basal media (Gibco/LifeTechnologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum(Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units permL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence. Cells were seeded into96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/wellfor use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

NHDF cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

Treatment with antisense compounds:

When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired oligonucleotide at a final concentration of150 nM. After 4 hours of treatment, the medium was replaced with freshmedium. Cells were harvested 16 hours after oligonucleotide treatment.

Example 10

Analysis of Oligonucleotide Inhibition of SRA Expression

Antisense modulation of SRA or SRA expression can be assayed in avariety of ways known in the art. For example, SRA mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. Methods of RNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons,Inc., 1993. Northern blot analysis is routine in the art and is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996.Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions. Other methods of PCR are alsoknown in the art.

It is presently believed that SRA is not translated to a protein.However, if appropriate, protein levels can be quantitated in a varietyof ways well known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), ELISA or fluorescence-activated cell sorting(FACS). Methods for preparation of polyclonal antisera are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997.Preparation of monoclonal antibodies is taught in, for example, Ausubel,F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp.11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 11

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996,42, 1758-1764. Other methods for poly(A)+ mRNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Briefly, for cells grown on 96-well plates, growth medium was removedfrom the cells and each well was washed with 200 μL cold PBS. 60 μLlysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40,20 mM vanadyl-ribonucleoside complex) was added to each well, the platewas gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the platewas blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C. was added to each well, the plate was incubated on a90° C. hot plate for 5 minutes, and the eluate was then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Example 12

Total RNA Isolation

Total mRNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 100 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 100 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and the vacuumagain applied for 15 seconds. 1 mL of Buffer RPE was then added to eachwell of the RNEASY 96™ plate and the vacuum applied for a period of 15seconds. The Buffer RPE wash was then repeated and the vacuum wasapplied for an additional 10 minutes. The plate was then removed fromthe QIAVAC™ manifold and blotted dry on paper towels. The plate was thenre-attached to the QIAVAC™ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA was then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step was repeated with an additional60 μL water.

Example 13

Real-time Quantitative PCR Analysis of SRA mRNA Levels

Quantitation of SRA mRNA levels was determined by realtime quantitativePCR using the ABI PRISM™ 7700 Sequence Detection System (PE-AppliedBiosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCR,in which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., JOE or FAM, obtained from either Operon Technologies Inc.,Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) isattached to the 5' end of the probe and a quencher dye (e.g., TAMRA,obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 3' end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3' quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5'-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular (six-second) intervals bylaser optics built into the ABI PRISM™ 7700 Sequence Detection System.In each assay, a series of parallel reactions containing serialdilutions of mRNA from untreated control samples generates a standardcurve that is used to quantitate the percent inhibition after antisenseoligonucleotide treatment of test samples.

PCR reagents were obtained from PE-Applied Biosystems, Foster City,Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail(1× TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of dATP, dCTP and dGTP,600 μM of dUTP, 100 nM each of forward primer, reverse primer, andprobe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLpoly(A) mRNA solution. The RT reaction was carried out by incubation for30 minutes at 48° C. Following a 10 minute incubation at 95° C. toactivate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension). SRA probes and primers were designedto hybridize to a consensus region of the four human SRA sequencesagainst which antisense oligonucleotides were designed, incorporatedherein as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4).

For SRA the PCR primers were:

forward primer: GTGGCCACACAAGGAAGCA (SEQ ID NO: 5) reverse primer:CTTTCCTCCAGCCCACTGTTC (SEQ ID NO: 6) and the PCR probe was:FAM-ATGACATCAGCCGACGCCTGGA-TAMRA (SEQ ID NO: 7) where FAM (PE-AppliedBiosystems, Foster City, Calif.) is the fluorescent reporter dye) andTAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

For GAPDH the PCR primers were:

forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 8) reverse primer:GAAGATGGTGATGGGATTTC (SEQ ID NO: 9)and the PCR probe was: 5'JOE-CAAGCTTCCCGTTCTCAGCC- TAMRA 3' (SEQ ID NO: 10) where JOE (PE-AppliedBiosystems, Foster City, Calif.) is the fluorescent reporter dye) andTAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

Example 14

Northern Blot Analysis of SRA mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST "B" Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST "B" Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.).

Membranes were probed using QUICKHYB™ hybridization solution(Stratagene, La Jolla, Calif.) using manufacturer's recommendations forstringent conditions with a SRA specific probe prepared by PCR using theforward primer GTGGCCACACAAGGAAGCA (SEQ ID NO: 5) and the reverse primerCTTTCCTCCAGCCCACTGTTC (SEQ ID NO: 6). To normalize for variations inloading and transfer efficiency membranes were stripped and probed forglyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, PaloAlto, Calif.). Hybridized membranes were visualized and quantitatedusing a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (MolecularDynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels inuntreated controls.

Example 15

Antisense Inhibition of SRA mRNA--Phosphorothioate Oligodeoxynucleotides

In accordance with the present invention, a series of oligonucleotideswere designed to target different regions of four sequences encodinghuman SRA RNA, incorporated herein as SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3 and SEQ ID NO: 4). The oligonucleotides are shown in Table 1.Target sites are indicated by nucleotide numbers on the target sequenceto which the oligonucleotide binds. All compounds in Table 1 areoligodeoxynucleotides with phosphorothioate backbones (internucleosidelinkages) throughout. The compounds were analyzed for effect on SRA mRNAlevels by quantitative real-time PCR as described in other examplesherein. Data are averages from two experiments. If present, "N.D."indicates "no data".

                  TABLE 1                                                         ______________________________________                                        Inhibition of SRA mRNA levels by phosphorothioate                              oligodeoxynucleotides                                                                  TAR-                                                                                                             GET                                 (SEQ TARGET  % SEQ ID                                                        ISIS# ID) SITE SEQUENCE Inhibition NO.                                      ______________________________________                                        30138 1      1        cgagcgcctgggtccgcc                                                                        52     11                                     30139 1 18 accctcttggtaagcagc 0 12                                            30140 1 77 gaggcccaggagaagtct 38 13                                           30141 1 114 ggagccttacttgaagga 46 14                                          30142 1 139 accactccccacaggtgg 0 15                                           30143 1 148 agaggcaggaccactccc 8 16                                           30144 1 158 gctccacgccagaggcag 51 17                                          30145 1 168 aaacttgtgggctccacg 82 18                                          30146 1 178 ctcgactgggaaacttgt 37 19                                          30147 1 188 gagcctcagactcgactg 35 20                                          30148 1 199 ctccatcagtcgagcctc 0 21                                           30149 1 208 cagcacatcctccatcag 39 22                                          30150 1 220 ttccaaaggtctcagcac 51 23                                          30151 1 229 caatgcctgttccaaagg 9 24                                           30152 1 240 cggcagtcttccaatgcc 0 25                                           30153 1 250 tgtgtggccacggcagtc 53 26                                          30154 1 261 acctgcttccttgtgtgg 68 27                                          30155 1 272 tgtcatcacatacctgct 24 28                                          30156 1 282 cgtcggctgatgtcatca 39 29                                          30157 1 292 cagtgccaggcgtcggct 34 30                                          30158 1 303 tgttcctgcagcagtgcc 61 31                                          30159 1 314 ctccagcccactgttcct 0 32                                           30160 1 325 tgacaactttcctccagc 85 33                                          30161 1 336 tttacaggtattgacaac 0 34                                           30162 1 347 ccattctcttctttacag 40 35                                          30163 1 359 gcaccagtagagccattc 70 36                                          30164 1 370 tgaaagctcttgcaccag 36 37                                          30165 1 382 ccaccggtggcttgaaag 15 38                                          30166 1 394 atctgctgcgtcccaccg 10 39                                          30167 1 403 gtggatgtcatctgctgc 0 40                                           30168 1 414 atgagggagcggtggatg 0 41                                           30169 1 425 catggtcaaccatgaggg 0 42                                           30170 1 438 ctgacctcagtcacatgg 0 43                                           30171 1 450 accatccactgactgacc 0 44                                           30172 1 462 cttttaactcctaccatc 57 45                                          30173 1 478 cttttctgcaattaatct 39 46                                          30174 1 491 aaaacagactcctctttt 46 47                                          30175 1 507 ttggctgcctcctctgaa 42 48                                          30176 1 542 ggttcttctcagctgtgg 51 49                                          30177 1 585 ggaaccgaggattatgaa 32 50                                          30218 2 1 ccgcgttccttgttgccg 6 51                                             30219 2 7 ttccagccgcgttccttg 0 52                                             30220 2 22 aactgcggcgggtcgttc 0 53                                            30221 2 47 ggcctgggtctgcagccc 6 54                                            30222 2 59 cctgggtccgccggcctg 14 55                                           30223 2 75 tggtaagcagcgagcgcc 26 56                                           30224 2 139 ggcccaggagaagtctct 0 57                                           30225 2 167 tgaaggaggtggaggccc 0 58                                           30226 2 204 gaccactccccacaggtg 0 59                                           30227 2 238 actgggaaacttgtgggc 0 60                                           30229 2 273 tcagcacatcctccatca 3 61                                           30230 2 310 tggccacggcagtcttcc 48 62                                          30231 2 324 cctgcttccttgtgtggc 34 63                                          30232 2 345 gtcggctgatgtcatcac 0 64                                           30233 2 383 ctttcctccagcccactg 64 65                                          30236 2 456 ctgctgcgtcccaccggt 79 66                                          30237 2 471 agcggtggatgtcatctg 35 67                                          30238 2 492 tcacatggtcaaccatga 7 68                                           30239 2 525 ttttaactcctaccatcc 0 69                                           30240 2 540 tttctgcaattaatcttt 0 70                                           30241 2 561 cctctgaaaacagactcc 29 71                                          30242 2 581 tttctcttcattggctgc 62 72                                          30243 2 599 ctcagctgtggctgcaga 0 73                                           30244 2 615 gtatggtatggttcttct 6 74                                           30245 2 629 ctgctggaagcctggtat 23 75                                          30246 2 637 tatgaagcctgctggaag 36 76                                          30247 2 667 ggtgtccggtgagtctgg 50 77                                          30248 2 686 tctccaaggcataggaga 20 78                                          30249 2 696 tgacagaaggtctccaag 19 79                                          30250 2 705 ggagccaagtgacagaag 14 80                                          30251 2 720 ggtggtggtaagaaggga 0 81                                           30252 2 745 gggtcaggcccagtggga 0 82                                           30253 2 759 cttccctcataggtgggt 0 83                                           30254 2 779 ctctggcccaggtgggac 45 84                                          30255 2 792 acacatgaactccctctg 23 85                                          30256 2 808 atgcatgttatgagtaac 0 86                                           30257 2 835 tttcaccgcagagatgtt 0 87                                           30371 3 56 gagggtccatactaagcg 47 88                                           30372 3 103 gaggcccggaactccact 11 89                                          30373 3 140 ttccttgttgcctgcgga 28 90                                          30374 3 174 cgtatgagaactgcggcg 0 91                                           30375 3 284 aggcccaggagaagtctc 39 92                                          30376 3 361 acgccagaggcaggacca 45 93                                          30377 3 400 agtcgagcctcagactcg 28 94                                          30378 3 473 acatacctgcttccttgt 0 95                                           30379 3 492 ggcgtcggctgatgtcat 58 96                                          30380 3 509 ttcctgcagcagtgccag 55 97                                          30381 3 529 aactttcctccaqcccac 79 98                                          30382 3 547 ttctttacaggtattgac 0 99                                           30383 3 581 qcttgaaagctcttgcac 0 100                                          30384 3 601 tctgctgcqtcccaccgg 68 101                                         30385 3 620 gagggagcggtggatgtc 52 102                                         30386 3 640 tcagtcacatggtcaacc 50 103                                         30387 3 663 ctcctaccatccactgac 54 104                                         30388 3 683 ttctgcaattaatctttt 41 105                                         30389 3 702 ctgaaaacagactcctct 44 106                                         30390 3 720 cttcattggctgcctcct 80 107                                         30391 3 756 tggtatggttcttctcag 42 108                                         30392 3 772 tgctggaagcctggtatg 72 109                                         30393 3 791 aaccgaggattatgaagc 43 110                                         30394 3 827 ggcaccggaaggagatgg 57 111                                         30395 3 847 gacaccagagggactagc 27 112                                         30396 3 867 cagggagcagggcagtcg 65 113                                         30397 3 885 cagaggtttgcagataca 65 114                                         30398 3 903 tgaatggagaagggagaa 25 115                                         30399 3 922 ccagcagatcccttcctg 0 116                                          30400 3 944 gtaggcagtagtctgact 58 117                                         30401 3 977 ggatgcttgttcagtcta 0 118                                          30402 3 992 gctccgcccagcacagga 63 119                                         30403 3 1007 catccaaacacagctgct 61 120                                        30404 3 1027 ttctcatgctgaaatgaa 26 121                                        30309 4 269 cccggcttcacgtacagc 37 122                                         30310 4 288 ccagccgcgttccttgtt 31 123                                         30311 4 323 gtctgcagcccgtatgag 0 124                                          30312 4 339 gggtccgccggcctgggt 0 125                                          30313 4 359 ttggtaagcagcgagcgc 44 126                                         30314 4 448 aaggaggtggaggcccca 28 127                                         30315 4 462 gggagccttacttgaagg 12 128                                         30316 4 498 gccagaggcaggaccact 28 129                                         30317 4 549 atcctccatcagtcgagc 0 130                                          30318 4 566 tccaaaggtctcagcaca 74 131                                         30319 4 600 ccttgtgtggccacggca 57 132                                         30320 4 616 catcacatacctgcttcc 32 133                                         30321 4 635 qccaggcgtcggctgatg 62 134                                         30322 4 669 caactttcctccagccca 59 135                                         30323 4 703 ccagtagagccattctct 17 136                                         30324 4 738 tgctgcgtcccaccggtg 73 137                                         30328 4 739 gctgtggctgcagatttc 52 138                                         30325 4 773 acatggtcaaccatgagg 46 139                                         30326 4 807 tttaactcctaccatcca 11 140                                         30327 4 842 tctgaaaacagactcctc 1 141                                          30329 4 913 cctgctggaagcctggta 0 142                                          30330 4 962 atttgaggagatggtgtc 6 143                                          30331 4 997 ctttccttttgttgaggg 0 144                                          30332 4 1032 gtatctcaagggtgatac 40 145                                        30333 4 1067 ccccactcaatgtggttc 69 146                                      ______________________________________                                    

As shown in Table 1, SEQ ID NOs 11, 13, 14, 17, 18, 19, 20, 22, 23, 26,27, 29, 30, 31, 33, 35, 36, 37, 45, 46, 47, 48, 49, 50, 62, 63, 65, 66,67, 72, 76, 77, 84, 88, 92, 93, 96, 97, 98, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 113, 114, 117, 119, 120, 122, 123, 126,131, 132, 133, 134, 135, 137, 138, 139, 145 and 146 demonstrated atleast 30% inhibition of SRA mRNA levels in this assay and are thereforepreferred.

Example 16

Antisense Inhibition of SRA--Phosphorothioate 2'-MOE GapmerOligonucleotides

In accordance with the present invention, a second series ofoligonucleotides targeted to human SRA were synthesized. Theoligonucleotide sequences are shown in Table 2. Target sites areindicated by nucleotide numbers, as given in the target sequence, towhich the oligonucleotide binds.

All compounds in Table 2 are chimeric oligonucleotides ("gapmers") 18nucleotides in length, composed of a central "gap" region consisting often 2'-deoxynucleotides, which is flanked on both sides (5' and 3'directions) by four-nucleotide "wings". The wings are composed of2'-methoxyethyl (2'-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide.Cytidine residues in the 2'-MOE wings are 5-methylcytidines.

Data were obtained by real-time quantitative PCR as described in otherexamples herein and are averaged from two experiments. If present,"N.D." indicates "no data".

                  TABLE 2                                                         ______________________________________                                        Inhibition of SRA mRNA levels by chimeric phosphorothioate                      oligonucleotides having 2'-MOE wings and a deoxy gap                                  TAR-                                                                   GET                                                                           (SEQ TARGET  % SEQ ID                                                        ISIS# ID) SITE SEQUENCE Inhibition NO.                                      ______________________________________                                        30178 1      1        cgagcgcctgggtccgcc                                                                        54     11                                     39179 1 18 accctcttggtaagcagc 0 12                                            30180 1 77 gaggcccaggagaagtct 44 13                                           30181 1 114 ggagccttacttgaagga 38 14                                          30182 1 139 accactccccacaggtgg 49 15                                          30183 1 148 agaggcaggaccactccc 0 16                                           30184 1 158 gctccacgccagaggcag 44 17                                          30185 1 168 aaacttgtgggctccacg 48 18                                          30186 1 178 ctcgactgggaaacttgt 39 19                                          30187 1 188 gagcctcagactcgactg 24 20                                          30188 1 199 ctccatcagtcgagcctc 28 21                                          30189 1 208 cagcacatcctccatcag 0 22                                           30190 1 220 ttccaaaggtctcagcac 44 23                                          30191 1 229 caatgcctgttccaaagg 29 24                                          30192 1 240 cggcagtcttccaatgcc 0 25                                           30193 1 250 tgtgtggccacggcagtc 0 26                                           30194 1 261 acctgcttccttgtgtgg 0 27                                           30195 1 272 tgtcatcacatacctgct 0 28                                           30196 1 282 cgtcggctgatgtcatca 57 29                                          30197 1 292 cagtgccaggcgtcggct 39 30                                          30198 1 303 tgttcctgcagcagtgcc 0 31                                           30199 1 314 ctccagcccactgttcct 27 32                                          30200 1 325 tgacaactttcctccagc 18 33                                          30201 1 336 tttacaggtattgacaac 0 34                                           30202 1 347 ccattctcttctttacag 29 35                                          30203 1 359 gcaccagtagagccattc 22 36                                          30204 1 370 tgaaagctcttgcaccag 27 37                                          30205 1 382 ccaccggtggcttgaaag 39 38                                          30206 1 394 atctgctgcgtcccaccg 22 39                                          30207 1 403 gtggatgtcatctgctgc 63 40                                          30208 1 414 atgagggagcggtggatg 43 41                                          30209 1 425 catggtcaaccatgaggg 56 42                                          30210 1 438 ctgacctcagtcacatgg 41 43                                          30211 1 450 accatccactgactgacc 0 44                                           30212 1 462 cttttaactcctaccatc 42 45                                          30213 1 478 cttttctgcaattaatct 0 46                                           30214 1 491 aaaacagactcctctttt 31 47                                          30215 1 507 ttggctgcctcctctgaa 68 48                                          30216 1 542 ggttcttctcagctgtgg 0 49                                           30217 1 585 ggaaccgaggattatgaa 71 50                                          30258 2 1 ccgcgttccttgttgccg 53 51                                            30259 2 7 ttccagccgcgttccttg 0 52                                             30260 2 22 aactgcggcgggtcgttc 0 53                                            30261 2 47 ggcctgggtctgcagccc 37 54                                           30262 2 59 cctgggtccgccggcctg 27 55                                           30263 2 75 tggtaagcagcgagcgcc 0 56                                            30264 2 139 ggcccaggagaagtctct 43 57                                          30265 2 167 tgaaggaggtggaggccc 29 58                                          30266 2 204 gaccactccccacaggtg 28 59                                          30267 2 238 actgggaaacttgtgggc 7 60                                           30269 2 273 tcagcacatcctccatca 0 61                                           30270 2 310 tggccacggcagtcttcc 50 62                                          30271 2 324 cctgcttccttgtgtggc 59 63                                          30272 2 345 gtcggctgatgtcatcac 38 64                                          30273 2 383 ctttcctccagcccactg 0 65                                           30276 2 456 ctgctgcgtcccaccggt 83 66                                          30277 2 471 agcggtggatgtcatctg 67 67                                          30278 2 492 tcacatggtcaaccatga 62 68                                          30279 2 525 ttttaactcctaccatcc 0 69                                           30280 2 540 tttctgcaattaatcttt 18 70                                          30281 2 561 cctctgaaaacagactcc 3 71                                           30282 2 581 tttctcttcattggctgc 77 72                                          30283 2 599 ctcagctgtggctgcaga 55 73                                          30284 2 615 gtatggtatggttcttct 56 74                                          30285 2 629 ctgctggaagcctggtat 51 75                                          30286 2 637 tatgaagcctgctggaag 22 76                                          30287 2 667 ggtgtccggtgagtctgg 0 77                                           30288 2 686 tctccaaggcataggaga 0 78                                           30289 2 696 tgacagaaggtctccaag 73 79                                          30290 2 705 ggagccaagtgacagaag 27 80                                          30291 2 720 ggtggtggtaagaaggga 34 81                                          30292 2 745 gggtcaggcccagtggga 0 82                                           30293 2 759 cttccctcataggtgggt 51 83                                          30294 2 779 ctctggcccaggtgggac 50 84                                          30295 2 792 acacatgaactccctctg 41 85                                          30296 2 808 atgcatgttatgagtaac 70 86                                          30297 2 835 tttcaccgcagagatgtt 35 87                                          30407 3 56 gagggtccatactaagcg 70 88                                           30408 3 103 gaggcccggaactccact 53 89                                          30409 3 140 ttccttgttgcctgcgga 69 90                                          30410 3 174 cgtatgagaactgcggcg 88 91                                          30411 3 284 aggcccaggagaagtctc 79 92                                          30412 3 361 acgccagaggcaggacca 57 93                                          30413 3 400 agtcgagcctcagactcg 71 94                                          30414 3 473 acatacctgcttccttgt 22 95                                          30415 3 492 ggcgtcggctgatgtcat 73 96                                          30416 3 509 ttcctgcagcagtgccag 85 97                                          30417 3 529 aactttcctccagcccac 69 98                                          30418 3 547 ttctttacaggtattgac 70 99                                          30419 3 581 gcttgaaagctcttgcac 77 100                                         30420 3 601 tctgctgcgtcccaccgg 82 101                                         30421 3 620 gagggagcggtggatgtc 35 102                                         30422 3 640 tcagtcacatggtcaacc 78 103                                         30423 3 663 ctactaccatccactgac 56 104                                         30424 3 683 ttctgcaattaatctttt 67 105                                         30425 3 702 ctgaaaacagactcctct 76 106                                         30426 3 720 cttcattggctgcctcct 81 107                                         30427 3 756 tggtatggttcttctcag 90 108                                         30428 3 772 tgctggaagcctggtatg 88 109                                         30429 3 791 aaccgaggattatgaagc 78 110                                         30430 3 827 ggcaccggaaggagatgg 48 111                                         30431 3 847 gacaccagagggactagc 25 112                                         30432 3 867 cagggagcagggcagtcg 43 113                                         30433 3 885 cagaggtttgcagataca 83 114                                         30434 3 903 tgaatggagaagggagaa 56 115                                         30435 3 922 ccagcagatcccttcctg 32 116                                         30436 3 944 gtaggcagtagtctgact 75 117                                         30437 3 977 ggatgcttgttcagtcta 21 118                                         30438 3 992 gctccgcccagcacagga 79 119                                         30439 3 1007 catccaaacacagctgct 72 120                                        30440 3 1027 ttctcatgctgaaatgaa 62 121                                        30345 4 269 cccggcttcacgtacagc 57 122                                         30346 4 288 ccagccgcgttccttgtt 73 123                                         30347 4 323 gtctgcagcccgtatgag 36 124                                         30348 4 339 gggtccgccggcctgggt 23 125                                         30349 4 359 ttggtaagcagcgagcgc 83 126                                         30350 4 448 aaggaggtggaggcccca 52 127                                         30351 4 462 gggagccttacttgaagg 3 128                                          30352 4 498 gccagaggcaggaccact 82 129                                         30353 4 549 atcctccatcagtcgagc 0 130                                          30354 4 566 tccaaaggtctcagcaca 80 131                                         30355 4 600 ccttgtgtggccacggca 82 132                                         30356 4 616 catcacatacctgcttcc 35 133                                         30357 4 635 gccaggcgtcggctgatg 64 134                                         30358 4 669 caactttcctccagccca 41 135                                         30359 4 703 ccagtagagccattctct 70 136                                         30360 4 738 tgctgcgtcccaccggtg 87 137                                         30364 4 739 gctgtggctgcagatttc 76 138                                         30361 4 773 acatggtcaaccatgagg 87 139                                         30362 4 807 tttaactcctaccatcca 14 140                                         30363 4 842 tctgaaaacagactcctc 38 141                                         30365 4 913 cctgctggaagcctggta 74 142                                         30366 4 962 atttgaggagatggtgtc 56 143                                         30367 4 997 ctttccttttgttgaggg 60 144                                         30368 4 1032 gtatctcaagggtgatac 72 145                                        30369 4 1067 ccccactcaatgtggttc 81 146                                      ______________________________________                                    

As shown in Table 2, SEQ ID NOs 11, 13, 14, 15, 17, 18, 19, 23, 29, 30,38, 40, 41, 42, 43, 45, 47, 48, 50, 51, 54, 57, 62, 63, 64, 66, 67, 68,72, 73, 74, 75, 79, 81, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 113, 114, 115, 116, 117, 119, 120, 121, 122, 123, 124, 126, 127,129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 141, 142, 143, 144,145 and 146 demonstrated at least 30% inhibition of SRA expression inthis experiment and are therefore preferred.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 146                                      - - <210> SEQ ID NO 1                                                        <211> LENGTH: 626                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 1                                                         - - ggcggaccca ggcgctcgct gcttaccaag agggtagccg caccccagga tg -            #gatccccc     60                                                                 - - agagtccccg catcagagac ttctcctggg cctcccccaa tggggcctcc ac -            #ctccttca    120                                                                 - - agtaaggctc ccaggtcccc acctgtgggg agtggtcctg cctctggcgt gg -            #agcccaca    180                                                                 - - agtttcccag tcgagtctga ggctcgactg atggaggatg tgctgagacc tt -            #tggaacag    240                                                                 - - gcattggaag actgccgtgg ccacacaagg aagcaggtat gtgatgacat ca -            #gccgacgc    300                                                                 - - ctggcactgc tgcaggaaca gtgggctgga ggaaagttgt caatacctgt aa -            #agaagaga    360                                                                 - - atggctctac tggtgcaaga gctttcaagc caccggtggg acgcagcaga tg -            #acatccac    420                                                                 - - cgctccctca tggttgacca tgtgactgag gtcagtcagt ggatggtagg ag -            #ttaaaaga    480                                                                 - - ttaattgcag aaaagaggag tctgttttca gaggaggcag ccaatgaaga ga -            #aatctgca    540                                                                 - - gccacagctg agaagaacca taccatacca ggcttccagc aggcttcata at -            #cctcggtt    600                                                                 - - ccccagactc accggacacc atctcc          - #                  - #                 626                                                                     - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 859                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 2                                                         - - cggcaacaag gaacgcggct ggaacgaccc gccgcagttc tcatacgggc tg -             #cagaccca     60                                                                 - - ggccggcgga cccaggcgct cgctgcttac caagagggtc gccgcacccc ag -            #gatggatc    120                                                                 - - ccccagagtc cccgcatcag agacttctcc tgggcctccc ccaatggggc ct -            #ccacctcc    180                                                                 - - ttcaagtaag gctcccaggt ccccacctgt ggggagtggt cctgcctctg gc -            #gtggagcc    240                                                                 - - cacaagtttc ccagtcgagt ctgaggctcg actgatggag gatgtgctga ga -            #cctttgga    300                                                                 - - acaggcattg gaagactgcc gtggccacac aaggaagcag gtatgtgatg ac -            #atcagccg    360                                                                 - - acgcctggca ctgctgcagg aacagtgggc tggaggaaag ttgtcaatac ct -            #gtaaagaa    420                                                                 - - gagaatggct ctactggtgc aagagctttc aagccaccgg tgggacgcag ca -            #gatgacat    480                                                                 - - ccaccgctcc ctcatggttg accatgtgac tgaggtcagt cagtggatgg ta -            #ggagttaa    540                                                                 - - aagattaatt gcagaaaaga ggagtctgtt ttcagaggag gcagccaatg aa -            #gagaaatc    600                                                                 - - tgcagccaca gctgagaaga accataccat accaggcttc cagcaggctt ca -            #taatcctc    660                                                                 - - ggttccccag actcaccgga caccatctcc tatgccttgg agaccttctg tc -            #acttggct    720                                                                 - - cccttcttac caccaccaag actgtcccac tgggcctgac ccacctatga gg -            #gaagaagt    780                                                                 - - cccacctggg ccagagggag ttcatgtgtt actcataaca tgcatttcaa ta -            #aaaacatc    840                                                                 - - tctgcggtga aaaaaaaaa             - #                  - #                      - #859                                                                  - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 1086                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 3                                                         - - gtagcgaccg gcgctcagct ggcgtcgccc cggcctaggc tgggggcggt tg -             #cggcgctt     60                                                                 - - agtatggacc ctctgtctcc cccagcccca gtatcagcta acagtggagt tc -            #cgggcctc    120                                                                 - - gcttcacaca tccctcgcct ccgcaggcaa caaggaacgc ggctggaacg ac -            #ccgccgca    180                                                                 - - gttctcatac gggctgcaga cccaggccgg cggacccagg cgctcgctgc tt -            #accaagag    240                                                                 - - ggtagccgca ccccaggatg gatcccccag agtccccgca tcagagactt ct -            #cctgggcc    300                                                                 - - tcccccaatg gggcctccac ctccttcaag taaggctccc aggtccccac ct -            #gtggggag    360                                                                 - - tggtcctgcc tctggcgtgg agcccacaag tttcccagtc gagtctgagg ct -            #cgactgat    420                                                                 - - ggaggatgtg ctgagacctt tggaacaggc attggaagac tgccgtggcc ac -            #acaaggaa    480                                                                 - - gcaggtatgt gatgacatca gccgacgcct ggcactgctg caggaacagt gg -            #gctggagg    540                                                                 - - aaagttgtca atacctgtaa agaagagaat ggctctactg gtgcaagagc tt -            #tcaagcca    600                                                                 - - ccggtgggac gcagcagatg acatccaccg ctccctcatg gttgaccatg tg -            #actgaggt    660                                                                 - - cagtcagtgg atggtaggag ttaaaagatt aattgcagaa aagaggagtc tg -            #ttttcaga    720                                                                 - - ggaggcagcc aatgaagaga aatctgcagc cacagctgag aagaaccata cc -            #ataccagg    780                                                                 - - cttccagcag gcttcataat cctcggttcc ccagactcac cggacaccat ct -            #ccttccgg    840                                                                 - - tgccaagcta gtccctctgg tgtcctcgac tgccctgctc cctgtgtatc tg -            #caaacctc    900                                                                 - - tgttctccct tctccattca tcaggaaggg atctgctggg taaagtcaga ct -            #actgccta    960                                                                 - - ccactttttc ccaaagtaga ctgaacaagc atcctgtgct gggcggagca gc -            #tgtgtttg   1020                                                                 - - gatggtttca tttcagcatg agaacagact caaatagaac gggccggaat tc -            #cgccgata   1080                                                                 - - ctgacg                 - #                  - #                  -     #         1086                                                                  - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 1123                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 4                                                         - - actcctggag cccgtcagta tcggcggccc tcatcacgtc cccacactgg ct -             #gtacagga     60                                                                 - - tcttgccatc ccccactcgg tcaaacagct cgaaggcctc cttgaactcc tc -            #cagctggt    120                                                                 - - ccttgttaaa ctcgatcacc actttggaga gatcgactgg aggctcctgg gt -            #tttctgag    180                                                                 - - gggcctgggg gacagctggc tcagctttgg tcttggctgg aggagcccct gc -            #tgctgctg    240                                                                 - - gcttagcagc aggtttggag atggaggagc tgtacgtgaa gccgggcaac aa -            #ggaacgcg    300                                                                 - - gctggaacga cccgccgcag ttctcatacg ggctgcagac ccaggccggc gg -            #acccaggc    360                                                                 - - gctcgctgct taccaagagg gtagccgcac cccaggatgg atcccccaga gt -            #ccccgcat    420                                                                 - - cagagacttc tcctgggcct cccccaatgg ggcctccacc tccttcaagt aa -            #ggctccca    480                                                                 - - ggtccccacc tgtggggagt ggtcctgcct ctggcgtgga gcccacaagt tt -            #cccagtcg    540                                                                 - - agtctgaggc tcgactgatg gaggatgtgc tgagaccttt ggaacaggca tt -            #ggaagact    600                                                                 - - gccgtggcca cacaaggaag caggtatgtg atgacatcag ccgacgcctg gc -            #actgctgc    660                                                                 - - aggaacagtg ggctggagga aagttgtcaa tacctgtaaa gaagagaatg gc -            #tctactgg    720                                                                 - - tgcaagagct ttcaagccac cggtgggacg cagcagatga catccaccgc tc -            #cctcatgg    780                                                                 - - ttgaccatgt gactgaggtc agtcagtgga tggtaggagt taaaagatta at -            #tgcagaaa    840                                                                 - - agaggagtct gttttcagag gaggcagcca atgaagagaa atctgcagcc ac -            #agctgaga    900                                                                 - - agaaccatac cataccaggc ttccagcagg cttcataatc ctcggttccc ca -            #gactcacc    960                                                                 - - ggacaccatc tcctcaaata gaacggggag acttttccct caacaaaagg aa -            #agacagtc   1020                                                                 - - ctatttgcac tgtatcaccc ttgagatact actgttacag agattagaac ca -            #cattgagt   1080                                                                 - - ggggttttct gtgtaaatcg aaggagaaaa agaccagatt act    - #                     112 - #3                                                                    - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 19                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR Primer                                            - - <400> SEQUENCE: 5                                                         - - gtggccacac aaggaagca             - #                  - #                      - # 19                                                                   - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR Primer                                            - - <400> SEQUENCE: 6                                                         - - ctttcctcca gcccactgtt c           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR Probe                                             - - <400> SEQUENCE: 7                                                         - - atgacatcag ccgacgcctg ga           - #                  - #                     22                                                                      - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 19                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR Primer                                            - - <400> SEQUENCE: 8                                                         - - gaaggtgaag gtcggagtc             - #                  - #                      - # 19                                                                   - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR Primer                                            - - <400> SEQUENCE: 9                                                         - - gaagatggtg atgggatttc            - #                  - #                      - # 20                                                                   - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: PCR Probe                                             - - <400> SEQUENCE: 10                                                        - - caagcttccc gttctcagcc            - #                  - #                      - # 20                                                                   - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 11                                                        - - cgagcgcctg ggtccgcc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 12                                                        - - accctcttgg taagcagc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 13                                                        - - gaggcccagg agaagtct             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 14                                                        - - ggagccttac ttgaagga             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 15                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 15                                                        - - accactcccc acaggtgg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 16                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 16                                                        - - agaggcagga ccactccc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 17                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 17                                                        - - gctccacgcc agaggcag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 18                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 18                                                        - - aaacttgtgg gctccacg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 19                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 19                                                        - - ctcgactggg aaacttgt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 20                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 20                                                        - - gagcctcaga ctcgactg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 21                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 21                                                        - - ctccatcagt cgagcctc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 22                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 22                                                        - - cagcacatcc tccatcag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 23                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 23                                                        - - ttccaaaggt ctcagcac             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 24                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 24                                                        - - caatgcctgt tccaaagg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 25                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 25                                                        - - cggcagtctt ccaatgcc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 26                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 26                                                        - - tgtgtggcca cggcagtc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 27                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 27                                                        - - acctgcttcc ttgtgtgg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 28                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 28                                                        - - tgtcatcaca tacctgct             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 29                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 29                                                        - - cgtcggctga tgtcatca             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 30                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 30                                                        - - cagtgccagg cgtcggct             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 31                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 31                                                        - - tgttcctgca gcagtgcc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 32                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 32                                                        - - ctccagccca ctgttcct             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 33                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 33                                                        - - tgacaacttt cctccagc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 34                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 34                                                        - - tttacaggta ttgacaac             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 35                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 35                                                        - - ccattctctt ctttacag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 36                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 36                                                        - - gcaccagtag agccattc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 37                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 37                                                        - - tgaaagctct tgcaccag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 38                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 38                                                        - - ccaccggtgg cttgaaag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 39                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 39                                                        - - atctgctgcg tcccaccg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 40                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 40                                                        - - gtggatgtca tctgctgc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 41                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 41                                                        - - atgagggagc ggtggatg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 42                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 42                                                        - - catggtcaac catgaggg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 43                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 43                                                        - - ctgacctcag tcacatgg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 44                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 44                                                        - - accatccact gactgacc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 45                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 45                                                        - - cttttaactc ctaccatc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 46                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 46                                                        - - cttttctgca attaatct             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 47                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 47                                                        - - aaaacagact cctctttt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 48                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 48                                                        - - ttggctgcct cctctgaa             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 49                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 49                                                        - - ggttcttctc agctgtgg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 50                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 50                                                        - - ggaaccgagg attatgaa             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 51                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 51                                                        - - ccgcgttcct tgttgccg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 52                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 52                                                        - - ttccagccgc gttccttg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 53                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 53                                                        - - aactgcggcg ggtcgttc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 54                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 54                                                        - - ggcctgggtc tgcagccc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 55                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 55                                                        - - cctgggtccg ccggcctg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 56                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 56                                                        - - tggtaagcag cgagcgcc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 57                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 57                                                        - - ggcccaggag aagtctct             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 58                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 58                                                        - - tgaaggaggt ggaggccc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 59                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 59                                                        - - gaccactccc cacaggtg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 60                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 60                                                        - - actgggaaac ttgtgggc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 61                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 61                                                        - - tcagcacatc ctccatca             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 62                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 62                                                        - - tggccacggc agtcttcc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 63                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 63                                                        - - cctgcttcct tgtgtggc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 64                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 64                                                        - - gtcggctgat gtcatcac             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 65                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 65                                                        - - ctttcctcca gcccactg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 66                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 66                                                        - - ctgctgcgtc ccaccggt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 67                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 67                                                        - - agcggtggat gtcatctg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 68                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 68                                                        - - tcacatggtc aaccatga             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 69                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 69                                                        - - ttttaactcc taccatcc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 70                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 70                                                        - - tttctgcaat taatcttt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 71                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 71                                                        - - cctctgaaaa cagactcc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 72                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 72                                                        - - tttctcttca ttggctgc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 73                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 73                                                        - - ctcagctgtg gctgcaga             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 74                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 74                                                        - - gtatggtatg gttcttct             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 75                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 75                                                        - - ctgctggaag cctggtat             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 76                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 76                                                        - - tatgaagcct gctggaag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 77                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 77                                                        - - ggtgtccggt gagtctgg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 78                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 78                                                        - - tctccaaggc ataggaga             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 79                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 79                                                        - - tgacagaagg tctccaag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 80                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 80                                                        - - ggagccaagt gacagaag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 81                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 81                                                        - - ggtggtggta agaaggga             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 82                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 82                                                        - - gggtcaggcc cagtggga             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 83                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 83                                                        - - cttccctcat aggtgggt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 84                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 84                                                        - - ctctggccca ggtgggac             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 85                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 85                                                        - - acacatgaac tccctctg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 86                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 86                                                        - - atgcatgtta tgagtaac             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 87                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 87                                                        - - tttcaccgca gagatgtt             - #                  - #                      - #   18                                                                  - -  - - <210> SEQ ID NO 88                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 88                                                        - - gagggtccat actaagcg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 89                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 89                                                        - - gaggcccgga actccact             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 90                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 90                                                        - - ttccttgttg cctgcgga             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 91                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 91                                                        - - cgtatgagaa ctgcggcg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 92                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 92                                                        - - aggcccagga gaagtctc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 93                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 93                                                        - - acgccagagg caggacca             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 94                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 94                                                        - - agtcgagcct cagactcg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 95                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 95                                                        - - acatacctgc ttccttgt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 96                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 96                                                        - - ggcgtcggct gatgtcat             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 97                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 97                                                        - - ttcctgcagc agtgccag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 98                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 98                                                        - - aactttcctc cagcccac             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 99                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 99                                                        - - ttctttacag gtattgac             - #                  - #                      - # 18                                                                    - -  - - <210> SEQ ID NO 100                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 100                                                       - - gcttgaaagc tcttgcac             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 101                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 101                                                       - - tctgctgcgt cccaccgg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 102                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 102                                                       - - gagggagcgg tggatgtc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 103                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 103                                                       - - tcagtcacat ggtcaacc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 104                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 104                                                       - - ctcctaccat ccactgac             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 105                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 105                                                       - - ttctgcaatt aatctttt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 106                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 106                                                       - - ctgaaaacag actcctct             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 107                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 107                                                       - - cttcattggc tgcctcct             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 108                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 108                                                       - - tggtatggtt cttctcag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 109                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 109                                                       - - tgctggaagc ctggtatg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 110                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 110                                                       - - aaccgaggat tatgaagc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 111                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 111                                                       - - ggcaccggaa ggagatgg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 112                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 112                                                       - - gacaccagag ggactagc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 113                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 113                                                       - - cagggagcag ggcagtcg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 114                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 114                                                       - - cagaggtttg cagataca             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 115                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 115                                                       - - tgaatggaga agggagaa             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 116                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 116                                                       - - ccagcagatc ccttcctg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 117                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 117                                                       - - gtaggcagta gtctgact             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 118                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 118                                                       - - ggatgcttgt tcagtcta             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 119                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 119                                                       - - gctccgccca gcacagga             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 120                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 120                                                       - - catccaaaca cagctgct             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 121                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 121                                                       - - ttctcatgct gaaatgaa             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 122                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 122                                                       - - cccggcttca cgtacagc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 123                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 123                                                       - - ccagccgcgt tccttgtt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 124                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 124                                                       - - gtctgcagcc cgtatgag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 125                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 125                                                       - - gggtccgccg gcctgggt             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 126                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 126                                                       - - ttggtaagca gcgagcgc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 127                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 127                                                       - - aaggaggtgg aggcccca             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 128                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 128                                                       - - gggagcctta cttgaagg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 129                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 129                                                       - - gccagaggca ggaccact             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 130                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 130                                                       - - atcctccatc agtcgagc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 131                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 131                                                       - - tccaaaggtc tcagcaca             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 132                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 132                                                       - - ccttgtgtgg ccacggca             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 133                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 133                                                       - - catcacatac ctgcttcc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 134                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 134                                                       - - gccaggcgtc ggctgatg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 135                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 135                                                       - - caactttcct ccagccca             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 136                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 136                                                       - - ccagtagagc cattctct             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 137                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 137                                                       - - tgctgcgtcc caccggtg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 138                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 138                                                       - - gctgtggctg cagatttc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 139                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 139                                                       - - acatggtcaa ccatgagg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 140                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 140                                                       - - tttaactcct accatcca             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 141                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 141                                                       - - tctgaaaaca gactcctc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 142                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 142                                                       - - cctgctggaa gcctggta             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 143                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 143                                                       - - atttgaggag atggtgtc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 144                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 144                                                       - - ctttcctttt gttgaggg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 145                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 145                                                       - - gtatctcaag ggtgatac             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 146                                                 <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Antisense Oligonucleotide                             - - <400> SEQUENCE: 146                                                       - - ccccactcaa tgtggttc             - #                  - #                      - #  18                                                                 __________________________________________________________________________

What is claimed is:
 1. An antisense compound 8 to 30 nucleobases inlength targeted to human SRA or a nucleic acid molecule encoding humanSRA, wherein said antisense compound specifically hybridizes with anddecreases the function or amount of human SRA.
 2. The antisense compoundof claim 1 which is an antisense oligonucleotide.
 3. The antisensecompound of claim 2 wherein the antisense oligonucleotide has a sequencecomprising SEQ ID NO: 11, 13, 14, 15, 17, 18, 19, 20, 22, 23, 26, 27,29, 30, 31, 33, 35, 36, 37, 38, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50,62, 63, 64, 65, 66, 67, 68, 72, 73, 74, 75, 76, 77, 79, 81, 83, 84, 85,86, 87, 51, 54, 57, 88, 89, 90, 91, 92, 93, 94, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 114, 115,116, 117, 119, 120, 121, 122, 123, 124, 126, 127, 129, 131, 132, 133,134, 135, 136, 137, 139, 141, 138, 142, 143, 144, 145 or
 146. 4. Theantisense compound of claim 2 wherein the antisense oligonucleotide hasa sequence comprising SEQ ID NO: 11, 13, 14, 17, 18, 19, 23, 29, 30, 45,47, 48, 50, 62, 63, 66, 67, 72, 84, 88, 92, 93, 96, 97, 98, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 114, 117, 119, 120,122, 123, 126, 131, 132, 133, 134, 135, 137, 139, 138, 145 or
 146. 5.The antisense compound of claim 2 wherein the antisense oligonucleotidecomprises at least one modified internucleoside linkage.
 6. Theantisense compound of claim 5 wherein the modified internucleosidelinkage is a phosphorothioate linkage.
 7. The antisense compound ofclaim 2 wherein the antisense oligonucleotide comprises at least onemodified sugar moiety.
 8. The antisense compound of claim 7 wherein themodified sugar moiety is a 2'-O-methoxyethyl sugar moiety.
 9. Theantisense compound of claim 2 wherein the antisense oligonucleotidecomprises at least one modified nucleobase.
 10. The antisense compoundof claim 9 wherein the modified nucleobase is a 5-methylcytosine. 11.The antisense compound of claim 2 wherein the antisense oligonucleotideis a chimeric oligonucleotide.
 12. A pharmaceutical compositioncomprising the antisense compound of claim 1 and a pharmaceuticallyacceptable carrier or diluent.
 13. The pharmaceutical composition ofclaim 12 further comprising a colloidal dispersion system.
 14. Thepharmaceutical composition of claim 12 wherein the antisense compound isan antisense oligonucleotide.
 15. A method of decreasing the function oramount of SRA in human cells or tissues in vitro comprising contactingsaid cells or tissues in vitro with the antisense compound of claim 1 sothat the function or amount of SRA is inhibited.